Capturing sequences adjacent to type IIs restriction sites for genomic library mapping

ABSTRACT

The present invention relates to novel methods for sequencing and mapping genetic markers in polynucleotide sequences using Type-IIs restriction endonucleases. The methods herein described result in the “capturing” and determination of specific oligonucleotide sequences located adjacent to Type-IIs restriction sites. The resulting sequences are useful as effective markers for use in genetic mapping, screening and manipulation.

This application is a continuation-in-part of U.S. application Ser. No.08/307,881, filed Sep. 16, 1994, which is hereby incorporated byreference in its entirety for all purposes.

Research leading to the present invention was funded in part by NIHgrant Nos. 5-F32-HG00105 and ROI HG00813-02, and the government may havecertain rights to the invention.

The present invention generally relates to novel methods for isolating,characterizing and mapping genetic markers in polynucleotide sequences.More particularly, the present invention provides methods for mappinggenetic material using Type-IIs restriction endonucleases. The methodsherein described result in the “capturing” and determination of specificoligonucleotide sequences located adjacent to Type-IIs restrictionsites. The resulting sequences are useful as effective markers for usein genetic mapping, screening and manipulation.

BACKGROUND OF THE INVENTION

The relationship between structure and function of macromolecules is offundamental importance in the understanding of biological systems. Theserelationships are important to understanding, for example, the functionsof enzymes, structural proteins and signalling proteins, ways in whichcells communicate with each other, as well as mechanisms of cellularcontrol and metabolic feedback.

Genetic information is critical in continuation of life processes. Lifeis substantially informationally based and its genetic content controlsthe growth and reproduction of the organism and its complements. Theamino acid sequences of polypeptides, which are critical features of allliving systems, are encoded by the genetic material of the cell.Further, the properties of these polypeptides, e.g., as enzymes,functional proteins, and structural proteins, are determined by thesequence of amino acids which make them up. As structure and functionare integrally related, many biological functions may be explained byelucidating the underlying structural features which provide thosefunctions, and these structures are determined by the underlying geneticinformation in the form of polynucleotide sequences. Further, inaddition to encoding polypeptides, polynucleotide sequences also can beinvolved in control and regulation of gene expression. It thereforefollows that the determination of the make-up of this geneticinformation has achieved significant scientific importance.

Physical maps of genomic DNA assist in establishing the relationshipbetween genetic loci and the DNA fragments which carry these loci in aclone library. Physical maps include “hard” maps which are overlappingcloned DNA fragments (“contigs”) ordered as they are found in the genomeof origin, and “soft” maps which consist of long range restrictionenzyme and cytogenetic maps (Stefton and Goodfellow, 1992). In thelatter case, the combination of rare cutting restriction endonucleases(e.g., NotI) and pulse gel electrophoresis allows for the large scalemapping of genomic DNAs. These methods provide a low resolution or topdown approach to genomic mapping.

A bottom up approach is exemplified by construction of contiguous or“contig” maps. Initial attempts to construct contig maps for the humangenome have been based upon ordering inserts cloned into cosmids. Morerecent studies have utilized yeast artificial chromosomes (YACs) whichallow for cloning larger inserts. The construction of contig mapsrequire that many clones be examined (4-5 genome equivalents) in orderto assure that sufficient overlap between clones is achieved. Currently,four approaches are used to identify overlapping sequences.

The first method is restriction enzyme fingerprinting. This methodinvolves the electrophoretic sizing of restriction enzyme generated DNAfragments for each clone and establishing a criterion for clone overlapbased on the similarity of fragment sets produced for each clone. Thesensitivity and specificity of this approach has been improved bylabelling of fragments using ligation, and end-filling techniques. Thedetection of repetitive sequence elements (e.g., [GT]_(n)) has also beenemployed to provide characteristic markers.

The second method generally employed in mapping applications is thebinary scoring method. This method involves the immobilization ofmembers of a clone library to filters and hybridization with sets ofoligonucleotide probes. Several mathematical models have been developedto avoid the need for large numbers of the probe sets which are designedto detect the overlap regions.

A third method is the Sequence Tagged Site (“STS”) method. This methodemploys PCR techniques and gel analysis to generate DNA products whoselengths characterize them as being related to common regions of sequencethat are present in overlapping clones. The sequence of the primarypairs and the characteristic distance between them provides sufficientinformation to establish a single copy landmark (SCL) which is analogousto single copy probes that are unique in the entire genome.

A fourth method uses cross-hybridizing libraries. This method involvesthe immobilization of two or more pools of cosmid libraries followed bycross-hybridization experiments between pairs of the libraries. Thiscross-hybridization demonstrates shared cloned sequences between thelibrary pairs. See, e.g., Kupfer, et al., (1995) Genomics 27:90-100.

Although each of these methods is capable of generating useful physicalmaps of genomic DNA, they each involve complex series of reaction stepsincluding multiple independent synthesis, labelling and detectionprocedures.

Traditional restriction endonuclease mapping techniques, i.e., asdescribed above, typically utilize restriction enzymerecognition/cleavage sites as genetic markers. These methods generallyemploy Type-II restriction endonucleases, e.g., EcoRI, HindIII andBamHI, which will typically recognize specific palindromic nucleotidesequences, or restriction sites, within the polynucleotide sequence tobe mapped, and cleave the sequence at that site. The restrictionfragments which result from the cleavage of separate fragments of thepolynucleotide (i.e., from a prior digestion) are then separated bysize. Overlap is shown where restriction fragments of the same sizeappear from Type-II endonuclease digestion of separate polynucleotidefragments.

Type-IIs endonucleases, on the other hand, generally recognizenon-palindromic sequences. Further, these endonucleases generally cleaveoutside of their recognition site thus producing overhangs of ambiguousbase pairs. Szybalski, 1985, Gene 40:169-173. Additionally, as a resultof their non-palindromic recognition sequences, the use of Type-IIsendonucleases will generate more markers per Kb than a similar Type-IIendonuclease, e.g., approximately twice as often.

The use of Type-IIs endonucleases in mapping genomic markers has beendescribed in, e.g., Brenner, et al., P.N.A.S. 86:8902-8906 (1989). Themethods described involved cleavage of genomic DNA with a Type-IIsendonuclease, followed by polymerization with a mixture of the fourdeoxynucleotides as well as one of the four specific fluorescentlylabelled dideoxynucleotides (ddA, ddT, ddG or ddC). Each successiveunpaired nucleotide within the overhang of the Type-IIs cleavage sitewould be filled by either a normal nucleotide or the labelleddideoxynucleotide. Where the latter occurred, polymerization stopped.Thus, the polymerization reaction yields an array of double strandedfluorescent DNA fragments of slightly different sizes. By reading thesize from smallest size to largest, in each of the nucleotide groups,one can determine the specific sequence of the overhang. However, thismethod can be time consuming and yields only the sequence of theoverhang region.

Oligonucleotide probes have long been used to detect complementarynucleic acid sequences in a nucleic acid of interest (the “target”nucleic acid). In some assay formats, the oligonucleotide probe istethered, i.e., by covalent attachment, to a solid support, and arraysof oligonucleotide probes immobilized on solid supports have been usedto detect specific nucleic acid sequences in a target nucleic acid. See,e.g., U.S. patent application Ser. No. 08/082,937, filed Jun. 25, 1993,which is incorporated herein by reference. Others have proposed the useof large numbers of oligonucleotide probes to provide the completenucleic acid sequence of a target nucleic acid but failed to provide anenabling method for using arrays of immobilized probes for this purpose.See U.S. Pat. Nos. 5,202,231 and 5,002,867.

The development of VLSIPS™ (Very Large Substrate Immobilized PolymerSynthesis) technology has provided methods for making very largecombinations of oligonucleotide probes in very small arrays. See U.S.Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and92/10092, each of which is incorporated herein by reference in itsentirety for all purposes. U.S. patent application Ser. No. 08/082,937,incorporated above, also describes methods for making arrays ofoligonucleotide probes that can be used to provide the complete sequenceof a target nucleic acid and to detect the presence of a nucleic acidcontaining a specific nucleotide sequence.

The construction of genetic linkage maps and the development of physicalmaps are essential steps on the pathway to determining the completenucleotide sequence of the human or other genomes. Present methods usedto construct these maps rely upon information obtained from a range oftechnologies including gel-based electrophoresis, hybridization,polymerase chain reaction (PCR) and chromosome banding. These methods,while providing useful mapping information, are very time consuming whenapplied to very large genome fragments or other nucleic acids. There istherefore a need to provide improved methods for the identification andcorrelation of genetic markers on a nucleic acid which can be used torapidly generate genomic maps. The present invention meets these andother needs.

SUMMARY OF THE INVENTION

The present invention provides methods for identifying specificoligonucleotide sequences using Type-IIs endonucleases in sequentialorder to capture the ambiguous sequences adjacent to the Type-IIsrecognition sites. These ambiguous sequences can then be probedsequentially with probes specific for the various combinations ofpossible ambiguous base pair sequences. By determining which probehybridizes with an ambiguous sequence, that sequence is thus determined.Further, because that sequence is adjacent to a specific Type-IIscleavage site that portion of the sequence is also known. Thiscontiguous sequence is useful as a marker sequence in mapping genomiclibraries.

In one embodiment, the present invention provides a method ofidentifying sequences in a polynucleotide sequence. The method comprisescleaving the polynucleotide sequence with a first type-IIs endonuclease.A first adapter sequence, having a recognition site for a secondtype-IIs endonuclease, is ligated to the polynucleotide sequence cleavedin the first cleaving step. The polynucleotide sequence resulting fromthe first ligating step, is cleaved with the second type-IIsendonuclease, and a second adapter sequence is ligated to thepolynucleotide sequence cleaved in the second cleaving step. Thesequence of nucleotides of the polynucleotide sequence between the firstand second adapter sequences is then determined.

In another embodiment, the present invention provides a method ofgenerating an ordered map of a library of genomic fragments. The methodccmprises identifying sequences in each of the genomic fragments in thelibrary, as described above. The identified sequences in each fragmentare compared with the sequences identified in each other fragment toobtain a level of correlation between each fragment and each otherfragment. The fragments are then ordered according to their level ofcorrelation.

In a further embodiment, the present invention provides a method ofidentifying polymorphisms in a target polynucleotide sequence. Themethod comprises identifying sequences in a wild-type polynucleotidesequence, according to the methods described above. The identifying stepis repeated on the target polynucleotide sequence. The differences inthe sequences identified in each of the identifying steps aredetermined, the differences being indicative of a polymorphism.

In still another embodiment, the present invention provides a method ofidentifying a source of a biological sample. The method comprisesidentifying a plurality of sequences in a polynucleotide sequencederived from the sample, according to the methods described herein. Theplurality of sequences identified in the identifying step are comparedwith a plurality of sequences identically identified from apolynucleotide derived from a known source. The identity of theplurality of sequences identified from the sample with the plurality ofsequences identified from the known source is indicative that the samplewas derived from the known source.

In an additional embodiment, the present invention provides a method ofdetermining a relative location of a target nucleotide sequence on apolynucleotide. The method comprises generating an ordered map of thepolynucleotide according to the methods described herein. Thepolynucleotide is fragmented. The fragment which includes the targetnucleotide sequence is then determined, and a marker on the fragment iscorrelated with a marker on the ordered map to identify the approximatelocation of the target nucleotide sequence on the polynucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of combinations of Type-IIs endonucleases usefulin the present invention. Gaps in the sequence illustrate the cleavagepattern of the first Type-IIs endonuclease, shown to the left, whereasarrows illustrate the cleavage points of the second Type-IIsendonuclease, shown to the right, when the recognition site for thatendonuclease is ligated to the first cleaved sequence. FIG. 1 also showsthe expected frequency of cleavage of the first Type-IIs endonuclease,the number of recognition sites in λ DNA, and the size of the sandwichedsequence.

FIG. 2 shows a schematic representation of an embodiment of the presentinvention for capturing Type-IIs restriction sites showing (1) a firstcleavage with EarI, (2) followed by a ligation to the 5′ overhang of afirst adapter sequence, (3) cleavage with HgaI, (4) ligation to secondadapter sequence followed by PCR amplification (5).

FIG. 3 shows a schematic representation of a preferred embodiment of thepresent invention using (1) a first cleavage with EarI followed by DNApolymerization of the overhang to yield a blunt end, (2) ligation toblunt end first adapter sequence, (3) melting off the unligated adapterstrand followed by DNA polymerization to extend dsDNA across the firstadapter strand, (4) cleavage with HgaI at the EarI recognition site, (5)ligation of second adapter sequence to target sequence, and (6)amplification/transcription of the captured target sequence.

FIG. 4 shows the combinatorial design for an oligonucleotide array usedto probe a four nucleotide captured ambiguous sequence. The probes uponthe array are 15mers having the sequence 3′C-T-G-C-G-w-x-y-z-C-T-T-C-T-C5′, where -w-x-y-z- are determined by the probe's position on the array.For example, the probe indicated by the darkened square on the arrayshown will have the w-x-y-z sequence of -A-T-G-C-.

FIG. 5 shows the predicted and actual fluorescent hybridization patternof captured sequences from λ DNA as described in Example 1 upon anoligonucleotide array probe having the combinatorial design of FIG. 4.Panel A shows the predicted hybridization pattern where the darkenedsquares indicate expected marker/probe hybridizations from capturedsequences from λ DNA cut with EarI and captured with HgaI bearingadapter sequences. The actual fluorescence of the hybridization is shownin panel B.

FIG. 6 shows a portion of known map of a yeast chromosomal library,illustrating the positions of each fragment of the library within yeastchromosome IV.

FIG. 7A shows a plot of correlation coefficient scores amonghybridization patterns of yeast chromosomal fragments when usingType-IIs and adjacent sequences as markers. FIG. 7B shows the predicted“correlation” scores for EarI captured marker sequences for fiftysimulated sequences from yeast chromosome III. The inner product scoresfor pair-wise comparison of the sequences is plotted versus the percentoverlap of the sequences. FIG. 7C shows the same simulated correlationusing BbsI captured marker sequences. FIG. 7D shows a simulatedcorrelation using HphI captured marker sequences.

FIGS. 8A, 8B and 8C show a schematic representation of theidentification of polymorphic markers, using the methods of the presentinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In general, the present invention provides novel methods for identifyingand characterizing sequence based nucleic acid markers as well as amethod for determining their presence. The methods may generally be usedfor generating maps for large, high molecular weight nucleic acids,i.e., for mapping short clones, cosmids, YACs, as well as in methods forgenetic mapping for entire genomes. Generally, the methods of thepresent invention involve the capturing of ambiguous nucleic acidsequence segments using sequential cleavage with restrictionendonucleases. In particular, the methods of the invention include afirst cleavage which leaves ambiguous sequences downstream from therecognition site of the cleavage enzyme. A second type-IIs recognitionsite is ligated to the target sequence, and a second cleavage,recognizing the second site, cleaves upstream from the first cleavagesite, within the first recognition site, resulting in short sequenceswhich contain the recognition site and an ambiguous sequence “captured”from the target sequence, between the two cleavage sites. Thecombination of the recognition site and the captured sequences areparticularly useful as genetic markers for genomic mapping applications.

In one embodiment, the methods of the present invention comprise the useof type-IIs endonucleases to capture sequences adjacent to the type-IIsrecognition site. These captured sequences then become effectivesequence based markers. More particularly, this method comprises firsttreating the polynucleotide sequence with a first Type-IIs endonucleasehaving a specific recognition site on the sequence, thereby cleaving thesequence. A first “adapter sequence” which comprises a second Type-IIsendonuclease recognition site is ligated to the cleaved sequence. Theresulting heterologous sequence thus has an ambiguous sequencesandwiched between two different Type-IIs endonuclease recognitionsites. This resulting sequence is then treated with a second Type-IIsendonuclease specific for the ligated recognition site, thereby cleavingthe sequence. A second adapter sequence is then ligated to this cleavedsequence. The sequence resulting from this ligation is then probed todetermine the sequence of the sandwiched, or “captured”, ambiguoussequence.

I. TYPE-IIS ENDONUCLEASES

Type-IIs endonucleases are generally commercially available and are wellknown in the art. Like their Type-II counterparts, Type-IIsendonucleases recognize specific sequences of nucleotide base pairswithin a double stranded polynucleotide sequence. Upon recognizing thatsequence, the endonuclease will cleave the polynucleotide sequence,generally leaving an overhang of one strand of the sequence, or “stickyend.”

Type-II endonucleases, however, generally require that the specificrecognition site be palindromic. That is, reading in the 5′ to 3′direction, the base pair sequence is the same for both strands of therecognition site. For example, the sequence

is the recognition site for the Type-II endonuclease EcoRI, where thearrows indicate the cleavage sites in each strand. This sequence ispalindromic in that both strands of the sequence, when read in the 5′ to3′ direction are the same.

The Type-IIs endonucleases, on the other hand, generally do not requirepalindromic recognition sequences. Additionally, these Type-IIsendonucleases also generally cleave outside of their recognition sites.For example, the Type-IIs endonuclease EarI recognizes and cleaves inthe following manner:

where the recognition sequence is -C-T-C-T-T-C-, N and n representcomplementary, ambiguous base pairs and the arrows indicate the cleavagesites in each strand. As the example illustrates, the recognitionsequence is non-palindromic, and the cleavage occurs outside of thatrecognition site. Because the cleavage occurs within an ambiguousportion of the polynucleotide sequence, it permits the capturing of theambiguous sequence up to the cleavage site, under the methods of thepresent invention.

Specific Type-IIs endonucleases which are useful in the presentinvention include, e.g., EarI, MnlI, PleI, AlwI, BbsI, BsaI, BsmAI,BspMI, Esp3I, HgaI, SapI, SfaNI, BbvI, BsmFI, FokI, BseRI, HphI andMboII. The activity of these Type-IIs endonucleases is illustrated inFIG. 1, which shows the cleavage and recognition patterns of theType-IIs endonucleases.

II. CAPTURING AMBIGUOUS SEQUENCES ADJACENT TO TYPE-IIS RESTRICTION SITES

A general schematic of the capturing of the ambiguous sequences is shownin FIGS. 2 and 3.

Treatment of the polynucleotide sequence sought to be mapped with aType-IIs endonuclease, results in a cleaved sequence having a number ofambiguous, or unknown, nucleotides adjacent to a Type-IIs endonucleaserecognition site within the target sequence. Additionally, within thisambiguous region, an overhang is created. The recognition site and theambiguous nucleotides are termed the “target sequence.” The overhang maybe 2, 3, 4 or 5 or more nucleotides in length while the ambiguoussequence may be from 4 to 9 or more nucleotides in length, both of whichwill depend upon the Type-IIs endonucleases used. Examples of specificType-IIs endonucleases for this first cleavage include BsmAI, EarI,MnlI, PleI, AlwI, BbsI, BsaI, BspMI, Esp3I, HgaI, SapI, SfaNI, BseRI,HphI and MboII. Again, these first Type-IIs endonucleases and theircleavage patterns are shown in FIG. 1, where the shaded region to theleft illustrates the recognition site of the first Type-IIsendonuclease, and gaps in the sequence illustrate the cleavage patternof the enzyme. Cleavage of high molecular weight DNA with EarI leaves anoverhang of three ambiguous base pairs, as shown in FIGS. 2 and 3,step 1. The recognition site of EarI is indicated by the bar. Thus, EarIcleavage of the target nucleic acid will produce a sequence having thefollowing cleavage end:

(SEQ ID NO: 2) -C-T-C-T-T-C-N- -G-A-G-A-A-G-n-n-n-n-

The overhanging bases are then filled in. This is preferably carried outby treatment of the target sequence with a DNA polymerase, such asKlenow fragment or T4 DNA polymerase, resulting in a blunt end sequenceas shown in FIG. 3; step 1. Alternatively however, the overhang may befilled by the hybridization of this overhang with an adapter sequencehaving an overhang complementary to that of the target sequence, asshown in FIG. 2, step 2. A tagging scheme, similar to this latter methodhas been described. See, D. R. Smith, PCR Meth. and Appl. 2:21-27(1992).

Following cleavage and fill in of the overhang portion, an adaptersequence is typically ligated to the cleavage end. The adapter sequencesdescribed in the present invention generally are specific polynucleotidesequences prepared for ligation to the target sequence. In preferredembodiments, these sequences will incorporate a second type-IIsrestriction site. Ligation of an adapter including a HgaI recognitionsite is shown in FIGS. 2 and 3, step 2. The adapter sequences aregenerally prepared by oligonucleotide synthesis methods generally wellknown in the art, such as the phosphoramidite or phosphotriester methodsdescribed in, e.g., Gait, oligonucleotide Synthesis: A PracticalApproach, IRL Press (1990).

An adapter sequence prepared to include a second type-IIs recognitionsite, for example, the HgaI recognition site 3′-C-G-C-A-G-S′ would beligated to the cleaved target sequence to provide a cleavage site on theother end of the ambiguous sequence. For example, ligation of the HgaIadapter to the target sequence would produce the following sequencehaving the cleavage pattern shown:

In addition to the Type-IIs recognition sites, preferred adaptersequences will also generally include PCR primers and/or promotersequences for in vitro transcription, thereby facilitating amplificationand labeling of the target sequence.

The method of ligation of the first adapter sequence to the targetsequence may be adapted depending upon the particular embodimentpracticed. For example, where ligation of the first adapter sequence isto the overhang of the target sequence, as shown in FIG. 2, step 2, theadapter sequence will generally comprise an overhang which iscomplementary to the overhang of the target sequence. For thisembodiment, a mixture of adapter sequences would generally be usedwherein all possible permutations of the overhang are present. Forexample, the number of specific probe sequences will typically be about4^(m) where m is the number of overhanging nucleotides. For example,where the target sequence after the first cleavage has a 4 base pairoverhang of ambiguous nucleotides, the mixture of sequences wouldtypically comprise adapters having upwards of 4⁴, or 256 differentoverhang sequences. Where the overhang in question includes greaternumbers of nucleotides, the adapters would generally be provided in twoor more separate mixtures to minimize potential ligation of the adapterswithin each mixture. For example, one set of adapters may incorporate apyrimidine nucleotide in a given position of the overhang for alladapters in the mixture whereas the other set will have a purinenucleotide in that position. As a result, ligation of the adapters toadapters in the same mixture will be substantially reduced. For longeroverhang sequences, it may often be desirable to provide additionalseparate mixtures of adapters. Ligation of the adapter sequence to thetarget sequence is then carried out using a DNA ligase according tomethods known in the art.

Where the overhang of the target sequence is filled in by Klenowfragment polymerization, as in FIG. 3, step 1, a blunt end adaptersequence is ligated to the target sequence. See, FIG. 3, step 2. Becausea blunt end ligation is used rather than an overhang, a mixture ofhybridizable sequences is unnecessary, and a single adapter sequence isused. Further, this method avoids any hybridization between theoverhangs in the mixture of adapter sequences.

Using this method, the polymerized target sequence will bephosphorylated on only the 5′ strand. Further, as the adapter will haveonly 3′ and 5′ hydroxyls for ligation, only the 3′ end of the adapterwill be ligated to the blunt, phosphorylated 5′ end of the targetsequence, leaving a gap in the other strand. The unligated strand of theadapter sequence may then be melted off and the remaining polynucleotideagain treated with DNA polymerase, e.g., Klenow or E. coli DNApolymerase, as shown in FIG. 3, step 3, resulting in a double-stranded,heterologous polynucleotide. This polynucleotide has the ambiguousnucleotide sequence sandwiched between the first Type-IIs endonucleaserecognition site (“site A”), and the second, ligated Type-IIsrecognition site (“site B”). One skilled in the art will recognize thatapproximately half of the adapter sequences will ligate to the targetsequence in an inverted orientation. However, this does not affect theresults of the methods of the present invention due to the inability ofthe second type-IIs enzyme to cleave the target sequence in those caseswhere the adapter is inverted. This is discussed in greater detail,below.

The polynucleotide resulting from ligation of this first adaptersequence to the target sequence is then treated with a second Type-IIendonuclease specific for the ligated recognition site B. This secondendonuclease treatment cleaves the remainder of the originalpolynucleotide from the target sequence. In preferred aspects, thesecond type-IIs endonuclease will be selected, or the second recognitionsite will be positioned within the adapter sequence, whereby thecleavage pattern of the second Type-IIs endonuclease results in thesecond cleavage substantially or entirely overlapping the firstrecognition site A, i.e., the cleavage of each strand is within oradjacent to the first recognition site (site A). FIG. 2, step 3, andFIG. 3, step 4 show the cleavage of the polynucleotide using HgaI (theHgaI recognition site is shown by the bar). Where the adapter sequenceis ligated in a reverse orientation, as previously noted, no cleavagewill occur within the first recognition site, as the recognition sitewill be at the distal end of the adapter sequence. Further, any primersequences present within this adapter will be inverted preventingsubsequent amplification. By selecting a second Type-IIs endonucleasedifferent from the first, recleavage of the first cleavage site isavoided. Selection of an appropriate type-IIs endonuclease for thesecond cleavage, and thus, the appropriate recognition site for thefirst adapter sequence, may often depend upon the first endonucleaseused, or as described above, the position of the recognition site withinthe adapter. In preferred aspects, the first and second type-IIsendonucleases are selected whereby the second endonuclease cleavesentirely within the first endonucleases recognition sequence. Examplesof Type-IIs endonucleases for the second cleavage generally includethose described above, and are typically selected from HgaI, BbvI,BspMI, BsmFI and FokI. Particularly preferred combinations of Type-IIsendonucleases for the first and second cleavages, as well as theircleavage patterns are shown in FIG. 1. Continuing with the previousexample, HgaI cleavage of the sample target sequence would produce thefollowing sequence having the ambiguous base pairs captured by the firstadapter sequence:

-C-T-C-T-T-C-N-N-N-N-G-C-G-T-C-           -G-n-n-n-n-C-G-C-A-G-

Depending upon the type-IIs endonucleases used in each step, thesequence of the overhang is known. For example, in the above example,the HgaI cleavage site for the second endonuclease is within the firstendonuclease's recognition site, e.g., the EarI site. An example of aknown overhang sequence is demonstrated in FIGS. 2 and 3, steps 4 and 5,respectively.

As noted, in the preferred aspects the second cleavage sitesubstantially or entirely overlaps the first recognition site A.Accordingly, the number of possible hybridizing sequences for thisligation step is rendered unique. The specific recognition site A of thefirst Type-IIs endonuclease is known. Thus, where the second cleavageoccurs entirely within the first recognition site A, only the uniquesequences hybridizing to that sequence would be used. On the other hand,where the second cleavage occurs to some extent outside of the firstrecognition site A, a mixture of specific adapter sequences hybridizableto all possible permutations of nucleotides outside of site A is used.For example, where cleavage incorporates one nucleotide outside of thefirst recognition site, the four variations to the known sequence arepossible and a mixture of adapter sequences hybridizable to all four isused (See, e.g., MnlI-HgaI enzyme pairing in FIG. 1). The number ofbases included in the second cleavage which fall outside the firstrecognition site is readily determinable from the endonucleases used.

As with the first adapter sequence, the second adapter sequence maycomprise a PCR primer sequence and/or a promoter sequence for in vitrotranscription.

The resulting target sequence will thus have the target sequence,specifically, an ambiguous sequence attached to a portion or all of thefirst recognition site, sandwiched or captured between the two adaptersequences. For example, the resulting target sequence will generallyhave the general sequence:

(Adapter sequence/A)−(Ambiguous sequence)−(B/Adapter sequence)

where A is a portion or all of the recognition site for the firstType-IIs endonuclease, and B is the recognition site for the secondType-IIs endonuclease. Again, applying the previous example, theresulting target sequence would appear as follows:

Adapter 2 -C-T-C-T-T-C-N-N-N-N-G-C-G-T-C- Adapter 1 Adapter2′-G-A-G-A-A-G-n-n-n-n-C-G-C-A-G- Adapter 1′

The sequence -C-T-C-T-T-C-N-N-N-N- is captured from the original targetsequence and sandwiched between the two adapter sequences.

Prior to probing, the target sequence will generally be amplified toincrease the detectability of the sequence. Amplification is generallycarried out by methods well known in the art. See FIGS. 2 and 3, steps 5and 6, respectively. For example, amplification may be performed by wayof polymerase chain reaction (PCR) using methods generally well known inthe art. See, e.g., Recombinant DNA Methodology, Wu, et al., ed.,Academic Press (1989), Sambrook, et al., Molecular Cloning: A LaboratoryManual (2nd ed.), vols. 1-3, Cold Spring Harbor Laboratory, (1989),Current Protocols in Molecular Biology, F. Ausubel, et al., ed., GreenePublishing and Wiley Interscience, New York (1987 and periodic updates).As described earlier, this amplification may be facilitated by theincorporation of specific primer sequences or complements within theadapters. Further, such amplification may also incorporate a label intothe amplified target sequence. In a preferred embodiment, the targetsequence may be amplified using an asymmetric PCR method whereby onlythe strand comprising the appropriate recognition site A is amplified.Asymmetric amplification is generally carried out by use of primer whichwill initiate amplification of the appropriate strand of the targetsequence, i.e., the target sequence.

The amplified target sequence may then be probed using specificoligonucleotide probes capable of hybridizing to the (A)-(ambiguoussequence)-(B) target sequence. As both the A and B sequences are set bythe capturing method and are known, the probes need only differ withrespect to the ambiguous portion of the sequence to be probed. Forexample, using the example sequence provided above, assuming that one isprobing with the top strand, e.g., the bottom strand was amplified byappropriate selection of primers, etc., the probes would generally havethe sequence C-T-C-T-T-C-n-n-n-n-G-C-G-T-C, where n denotes everypossible base at the particular position, e.g., A, T, G, C. Thepreparation of oligonucleotide probes is performed by methods generallyknown in the art. See, Gait, Oligonucleotide Synthesis: A PracticalApproach, IRL Press (1990). Additionally, these oligonucleotide probesmay be labelled, i.e., fluorescently or radioactively, so that probeswhich hybridize with target sequences can be detected. In preferredaspects, however, the probes will be immobilized, and it will be thetarget that is labelled. Labelling of the target sequence may be carriedout using known methods. For example, amplification of the targetsequence can incorporate a label into the amplified target sequence,e.g., by use of a labelled PCR primer or by incorporating a label duringin vitro transcription of either strand.

In the preferred embodiment of the present invention, the targetsequence is probed using an oligonucleotide array. Through the use ofthese oligonucleotide arrays, the specific hybridization of a targetsequence can be tested against a large number of individual probes in asingle reaction. Such oligonucleotide arrays employ a substrate,comprising positionally distinct sequence specific recognition reagents,such as polynucleotides, localized at high densities. A single array cancomprise a large number of individual probe sequences. Further, becausethe probes are in known positionally distinct orientations on thesubstrate, one need only examine the hybridization pattern of a targetoligonucleotide on the substrate to determine the sequence of the targetoligonucleotide. Use and preparation of these arrays for oligonucleotideprobing is generally described in PCT patent publication Nos. WO92/10092, WO 90/15070, U.S. patent application Ser. Nos. 08/143,312 and08/284,064. Each of these references is hereby incorporated by referencein its entirety for all purposes.

As noted, the target sequence will have the general sequence:

(Adapter sequence/A)−(N _(k))−(B/Adapter sequence)

where N_(k) denotes the ambiguous sequence of nucleotides of length k,and the nucleotide sequence of each adapter sequence is known and thesequence of sites A and B are known. Only the nucleotide sequence of theambiguous portion of the target sequence, N_(k) is not known. Thus, thenumber of probes required on the array substrate is generally related tothe number of ambiguous nucleotides in the target sequence. In oneembodiment, the number of potential sequences for an ambiguous sequenceis 4^(k), where k is the number of ambiguous bases within the sequence.For example, where there are four ambiguous nucleotides within thetarget sequence, the array would generally include about 4⁴ or 256 ormore separate probes, where each probe will include the generalsequence:

(A′)−(N′ _(k))−(B′)

where “A′” and “B′” are the complements to site-A and site-B of thetarget sequence, respectively and are constant throughout the array, and“N′_(k)” generally represents all potential sequences of the length ofthe ambiguous sequence of the target sequence. Thus, where the ambiguoussequence contains, e.g., 4 nucleotides, “N′_(k)” would typicallyinclude, for example, 4⁴ different sequences, at least one of which willhybridize with the target sequence. On an oligonucleotide array, this isaccomplished through a simple combinatorial array like that shown inFIG. 4. Typically, as the size of the ambiguous sequence increases, thenumber of probes on the array will also increase, e.g., where theambiguous sequence is 8 bases long, their will typically be about 4⁸ or65,536 probes on the array.

In the case of high molecular weight nucleic acids, the originalpolynucleotide sequence will generally comprise more than one and evenseveral specific Type-IIs endonuclease recognition/cleavage sites, e.g.,EarI sites. As a result, a number of ambiguous sequence segments will becaptured for a given polynucleotide. Upon probing with anoligonucleotide array, the sequence will hybridize with a number ofprobes which are complementary to all of the captured sequences,producing a distinctive hybridization pattern for the givenpolynucleotide sequence. The specific hybridization pattern of thetarget sequence upon the array will generally indicate the ambiguoussequences adjacent to all of the cleavage sites as was described above.

III. MAPPING GENOMIC LIBRARIES

A. Physical Maps

A further embodiment of the present invention provides a method for theordered mapping of genomic libraries. Typically, the term “genomiclibrary” is defined as a set of sequence fragments from a largerpolynucleotide fragment. Such larger fragments may be whole chromosomes,subsets thereof, plasmids, or other similar large polynucleotides.Specifically, the methods of the present invention are useful formapping high molecular weight polynucleotides including chromosomalfragments, cosmids and Yeast Artificial Chromosomes (YACs).

Mapping techniques typically involve the identification of specificgenetic markers on individual polynucleotide fragments from a genomiclibrary. Comparison of the presence and relative position of specificmarkers on fragments generated by different cleavage patterns allows forthe assembly of a contiguous genomic map, or “contig”.

Accordingly, in a particularly preferred aspect of the present inventionmethods of genomic mapping are provided utilizing the sequence capturingmethods already described. In particular, the methods of the presentinvention comprise identifying the Type-IIs and adjacent sequences(target sequences) on the individual fragments of a genomic libraryusing the methods described above. FIG. 6 shows a genomic map for aportion of a yeast chromosome library, showing the overlap between thevarious fragments of the library.

The individual fragments of the library are treated using the abovemethods to capture the Type-IIs restriction sites and their adjacentambiguous sequences. These captured sequences are then used as geneticmarkers, as described above, and a contig of the particular library maybe assembled. In the preferred aspects, the captured Type-IIs andadjacent sequences will be hybridized to specific positionally orientedprobes on the array. By determining the various probe sequences tohybridize with the captured sequences, these captured sequences arethereby determined.

The combination of these mapping techniques with oligonucleotide arraysprovides the capability of identifying a large number of genetic markerson a particular sequence. Typically, a genomic fragment will have morethan one, and even several Type-IIs restriction sites within itssequence. Thus, when probed with an oligonucleotide array, the capturedsequences from a particular genomic fragment will hybridize with anumber of probes on the array, producing a distinctive hybridizationpattern. Each hybridization pattern will generally comprisehybridization signals which correspond to each of the captured sequencemarkers in the fragment.

When repeated on separate fragments from the library, each fragment willgenerally produce a distinctive hybridization pattern, which reflectsthe sequences captured using the specific type-IIs capture method. Thesehybridization patterns may be compared with hybridization patterns fromdifferentially generated fragments. Where a specific marker is presentin both fragments, it is an indication of potential overlap between thefragments. Two fragments that share several of the same Type-IIssequences, e.g., overlapping fragments, will show similar hybridizationpatterns on the oligonucleotide array.

The greater the similarity or correlation between two fragments, thehigher the probability that these fragments share an overlappingsequence. By correlating the hybridization pattern of each fragment inthe library against each other fragment in the library, a singlecontiguous map of the particular library can be constructed.

In practice, each fragment is correlated to each other fragment, and acorrelation score is given based upon the number of probes whichcross-hybridize with the Type-IIs and adjacent sequences of both thefirst and second fragment. High scores indicates high overlap. Forexample, a perfect overlap, i.e., the comparison of two identicalsequences would produce a correlation score of 1. Similarly, sequencessharing no overlapping sequence would, ideally, produce a correlationscore of 0. However, in practice, sequences that do not overlap willgenerally have correlation scores above zero, due to potentialnon-specific hybridizations, e.g., single base mismatches, backgroundhybridization, duplicated sequences, which may provide some baselinecorrelations between otherwise unrelated fragments. As a result, acutoff may be established below which correlation scores are not used.The precise cutoff may vary depending upon the level of nonspecifichybridizations for the particular application. For example, by usingcapture methods that cut less frequently, and/or capture a greaternumber of sequences, the potential for duplicated markers issubstantially reduced, and the cutoff may be lower. Correlation scoresamong all of the fragments may then be extrapolated to provideapproximate percent overlap among the various fragments, and from thisdata, a contiguous map of the genomic library can be assembled (FIG.7A). Additionally, one of skill in the art will appreciate that a morestringent determination of cosmid overlap may be obtained by repeatingthe capture and correlation methods using a different enzyme system,thereby generating additional, different markers and overlap data.

The combined use of sequence based markers and oligonucleotide arrays,as described herein, provides a method for rapidly identifying a largenumber of genetic markers and mapping very large nucleic acid sequences,including, e.g., cosmids, chromosome fragments, YACs and the like.

The present invention also provides methods for diagnosing a geneticdisorder wherein said disorder is characterized by a mutation in asequence adjacent to a known Type-IIs endonuclease restriction siteusing the methods described above. Specifically, sequences adjacent toType-IIs restriction sites are captured and their sequence is determinedaccording to the methods described above. The determined sequence isthen compared to a “normal” sequence to identify mutations.

A. Genetic Linkage Mapping

Genetic linkage markers are defined as highly polymorphic sequenceswhich are uniformly distributed throughout a genome. In an additionalembodiment, the methods of the present invention are used to identifyand define these polymorphic markers. Because these markers areidentified and defined by their proximity to type-IIs restriction sites,they are referred to herein as restriction site sequence polymorphisms(“RSSPs”). In general, these RSSP markers are identified by comparingcaptured sequences among two genomes. The methods of the presentinvention may generally be used to identify these RSSPs in a number ofways. For example, a polymorphism within the recognition site of thetype-IIs endonuclease will result in the presence of a captured sequencein one genome where it is absent in the other. This is generally theresult where the polymorphism lies within the type-IIs recognition site,thereby eliminating the recognition site in the particular sequence,and, as a result, the ability to capture the adjacent sequences. It willbe appreciated that the inverse is also true, that a polymorphism mayaccount for the presence of a recognition site where one does not existin the wild type. Second, a polymorphism may be identified which lieswithin the captured ambiguous sequence. These polymorphisms willtypically be detected as a sequence difference between the comparedgenomes.

A wide variety of polymorphic markers may be identified for any givengenome, based upon the type-IIs enzymes used for the first and secondcleavages. For example, first cleavage enzymes which recognize distinctsequences will typically also define a number of distinct proximalpolymorphisms.

The above described methods may be further modified, for example, usingmethods similar to those reported by Nelson, et al., Nature Genetics(1993) 4:11-18. Nelson, et al. report the identification of polymorphicmarkers using a system of genetic mismatch scanning. In the method ofNelson, et al., the genomes to be compared, e.g., grandchild andgrandparent genomes, are first digested with an endonuclease whichproduces a 3′ overhang, i.e., PstI. One of the two genomes is methylatedat all GATC sites in the sequence (DAM+) while the other remainsunmethylated (DAM−). The genomic fragments from each group aredenatured, mixed with each other, and annealed, resulting in a mixtureof homohybrids and heterohybrids. In the homohybrids, both strands willbe either methylated or unmethylated, while in the heterohybrids, onestrand will be methylated. The mixture is then treated with nucleaseswhich will not cleave the hemimethylated nucleic acid duplexes, forexample DpmI and MboI. Next, the mixture is treated with a series ofmismatch repair enzymes, e.g., MutH, MutL and MutS, which introduce asingle strand nick on the duplexes which possess single base mismatches.The mixture is then incubated with ExoIII, a 3′ to 5′ exonuclease whichis specific for double stranded DNA, and which will degrade thepreviously digested homohybrids and the nicked strand of the mismatchedheterohybrids, from the 3′ side. Purification of the full dsDNA is thencarried out using methods known in the art, e.g., benzoylatednaphthoylated DEAE cellulose at high salt concentrations, which willbind ssDNA but not dsDNA. As a result, only the full-length, unaltered(perfectly matched) heterohybrids are purified. The recovered dsDNAfragments which indicate “identity by descent” (or “i.b.d.”) arelabelled and used to probe genomic DNA to identify sites of meioticrecombination.

An adaptation of the above method can be applied to the capture methodsof the present invention. In particular, the methods of the presentinvention pan be used to capture sequences in the region ofpolymorphisms in a particular polynucleotide sequence. FIGS. 8A, 8B and8C show a schematic representation of the steps used in practicing oneembodiment of this aspect of the present invention. Specifically, asubset of genomic DNA which is identified by the presence of a type-IIsrecognition site is amplified (FIG. 8A), DNA containing polymorphismswithin the amplified subset are isolated (FIG. 8B), and the sequencesadjacent to the type-IIs recognition site in the isolatedpolymorphism-containing sequences are identified and characterized (FIG.8C).

Initially, polynucleotides from different sources which are to becompared, e.g., grandparent-grandchild, etc., are treated identically inparallel systems. These polynucleotides are each cleaved with a firsttype-IIs endonuclease, as is described in substantial detail above. InFIG. 8A, step (a), for example, this first cleavage is shown usingBseR1. The specific Type-IIs enzyme used in this first cleavage mayagain vary depending upon the desired frequency of cleavage, the lengthof the target sequence, etc.

As previously described, a first adapter bearing, a second type-IIsendonuclease recognition site is ligated to the cleaved polynucleotides(FIG. 8A, step (b)). In the example of FIG. 8A, steps (a), (b) and (c),this recognition site is that of the type-IIs endonuclease FokI. Thepolynucleotides are then cleaved with an endonuclease which will cleaveupstream from the captured sequence and ligated first adapter, such as atype II endonuclease, e.g., HaeIII (sea FIG. 5A, step (d)). Typically,this second cleavage enzyme will be selected whereby it cleavage morefrequently than the first Type-IIs enzyme. A second adapter sequence maythen be ligated to this new cleavage site (FIG. 8A, step (e)). Theentire sequence, including the two adapter sequences is then typicallyamplified (FIG. 8A, step (f)). The amplification is facilitated inpreferred aspects by incorporating a primer sequence within the adaptersequences.

The amplified polynucleotides from each source is isolated (FIG. 8B,step (g)). The polynucleotide from one source is then methylated (FIG.8B, step (h)). Both the methylated polynucleotide from the first sourceand the unmethylated polynucleotide from the second source are mixedtogether, heated to denature duplex DNA, and reannealed (FIG. 8B, step(i)). This generally results in a mixture of hemimethylatedheterohybrids having one strand from each source, homohybrids ofunmethylated dsDNA and homohybrids of fully methylated dsDNA. At thispoint, unlike the method of Nelson, et al. (DpmI and MboI additions areomitted), the mixture is treated with the mismatch repair enzymes, e.g.,MutLSH, which will nick only hemimethylated, mismatched hybrids, leavingthe homohybrids and perfectly matched heterohybrids untouched (FIG. 8B,step (j)). The nicked DNA is then digested, as in Nelson, et al., withan exonuclease, e.g., ExoIII (FIG. 8B, step (k)). The mixture will thencontain dsDNA which is fully methylated, i.e., homohybrids of DNA fromone source, dsDNA which is unmethylated, i.e., homohybrids of DNA fromthe other source, heterohybrids of dsDNA from both sources, but whichare perfectly matched, i.e., contains no mismatches or polymorphisms,and ssDNA, i.e., the DNA which is left from the heterohybrid, mismatchedor polymorphic dsDNA. This ssDNA reflects the polymorphism and may thenbe purified from the dsDNA using the methods described in Nelson, etal., e.g., purification over benzoylated naphthoylated DEAE cellulose inhigh salt (FIG. 5C, step (l)).

The purified single stranded DNA is then reamplified to dsDNA usingmethods well know in the art, e.g., POI (FIG. 8C, step (m)). Theamplified DNA may then cleaved with a second type-IIs endonuclease whichrecognized the site incorporated into the first adapter sequence, asdescribed above (FIG. 8C, step (n)), followed by ligation of anotheradapter sequence to the cleavage end (FIG. 5C, step (o)). The capturedsequence thus identifies a polymorphism is which lies between thecaptured sequence and the upstream cleavage site. The captured sequencemay then be determined according to the methods described herein, e.g.,amplification, labelling and probing (FIG. 8C, step (p)).

IV. APPLICATIONS

The methods described herein are useful in a variety of applications.For example, as is described above, these methods can be used togenerate ordered physical maps of genomic libraries, as well as geneticlinkage maps which can be used in the study of genomes of varyingsources. The mapping of these genomes allows further study andmanipulation of the genome in diagnostic and therapeutic applicationse.g., gene therapy, diagnosis of genetic predispositions for particulardisorders and the like.

In addition to pure mapping applications, the methods of the presentinvention may also be used in other applications. In a preferredembodiment, the methods described herein are used in the identificationof the source of a particular sample. This application would includeforensic analysis to determine the origin of a particular tissue sample,such as analyzing blood or other evidence in criminal investigations,paternity investigations, etc. Additionally, these methods can also beused in other identification applications, for example, taxonomic studyof plants, animals, bacteria, fungi, viruses, etc. This taxonomic studyincludes determination of the particular identity of the species fromwhich a sample is derived, or the interrelatedness of samples from twoseparate species.

The various identification applications typically involve the capturingand identification of sequences adjacent specific type-IIs restrictionsites in a sample to be analyzed, according to the methods already:described. These sequences are then compared to sequences identicallycaptured and identified from a known source. Where sequences capturedfrom both the sample and the source are identical or highly similar, itis indicative that the sample was derived from the source. Where thesequences captured from the sample and known source share a large numberof identical sequences, it is indicative that the sample is related tothe known source. However, where the sample and source share few likesequences, it is indicative of a low probability of interrelation.

Precise levels of interrelation to establish a connection between sourceand sample, i.e., captured sequence homology, will typically beestablished based upon the interrelation which is being proved ordisproved, the identity of the known source, the precise method used,and the like. Establishing the level of interrelation is well within theordinary skill in the art. For example, in criminal investigations, ahigher level of homology between sample and known source sequences willlikely be required to establish the identity of the sample in question.Typically, in the criminal context, interrelation will be shown wherethere is greater than 95% captured sequence homology, preferably greaterthan 99% captured sequence homology, and more preferably, greater than99.9% captured sequence homology. For other identification applications,interrelation between sample and known source may be established by ashowing of, e.g., greater than 50% captured sequence homology, andtypically greater than 75% captured sequence homology, preferablygreater than 90% captured sequence homology, and more preferably greaterthan 95 to 99% captured sequence homology.

The level of interrelation will also typically vary depending upon theportion of a genome or nucleic acid sequence which is used forcomparison. For example, in attempting to identify a sample as beingderived from one member of a species as opposed to another member of thesame sperips, it will generally be desirable to capture sequences in aregion of the species' genetic material which displays a lower level ofhomology among the various members of the same species. This results ina higher probability of the captured sequences being specific to onemember of the species. The opposite can be true for taxonomic studies,i.e., to identify the genus and species of the sample. For example, itmay generally be desirable to select a portion of the genetic materialof the known genus or species which is highly conserved among members ofthe genus and/or species, thereby permitting identification of theparticular sample to that genus or species.

The present invention is further illustrated by the following examples.These examples are merely to illustrate aspects of the present inventionand are not intended as limitations of this invention. The methods usedgenerally employ commercially available reagents or reagents otherwiseknown in the art.

EXAMPLES Example 1 1. Digesting High Molecular Weight DNA with EarI

4 μg of λ DNA was treated with 4 units of EarI in 10 μl at 37° C. for 4hours. The reaction was then heated to 70° C. for 10 minutes. Cleavagewas verified by running 5 μl of the sample on an agarose gel todetermine complete cleavage. The remaining 5 μl was brought to 40 μl(final concentration of 50 ng/μl λ DNA).

2. Klenow Fill-in Reaction

4 μl of the digested A DNA was added to 0.5 μl of 10× Klenow Buffer, 0.5μl 2 mM dNTPs, and 0.05 μl of 0.25 units of Klenow fragment. Thereaction mixture was incubated for 20 minutes at 25° C., followed by 10minutes at 75° C. Similar results were also obtained using T4 DNApolymerase for the fill-in reaction.

3. Preparing Adapter Sequences

Two separate adapter sequences were prepared, adapter sequence 1 andadapter sequence 2. Adapter sequence 1 is used in the first ligationreaction whereas adapter 2 is used for the second. As each adapter andits ligation are somewhat different, they are addressed separately.

Double stranded adapter 1 comprising the second Type-IIs endonucleaserestriction site 3′ C-G-C-A-G- . . . 5′ and a T7 promoter sequence wasprepared by adding 10 μl each of 10 μM unphosphorylated T7 strand andits complement, heating the mixture to 95° C., then cooling over 20minutes to anneal the strands. The strands were prepared using DNAsynthesis methods generally well known in the art. The resulting mixturehad a final dsDNA adapter concentration of 5 μM.

Adapter 2 comprising the overhang complementary to that created by theHgaI digestion of the target sequence, as well as a T3 promoter sequencewas prepared by first creating the overhang region. A single strandedoligonucleotide of the sequence 3′ . . . -G-A-G-A-A 5′ was synthesizedon a single stranded T3 promoter sequence. The final concentration ofreagents is shown in parentheses. The 5′ end of this sequence was thenphosphorylated as follows: 10 μl of 10 μM the oligonucleotide (5 μM), 2μl of 10× kinase buffer (1×), 2 μl 10 mM ATP (1 mM), 5 μl water and 1 μlT4 polynucleotide kinase (10 units) were added. The reaction wasincubated at 37° C. for 60 minutes, then at 68° C. for 10 minutes andcooled.

To the T3/overhang ssDNA strand was added 10 μl of 10 μM appropriateantistrand and 3.33 μl of buffer. This mixture was heated to 95° C. andcooled over 20 minutes to anneal the two strands.

4. Ligation of First Adapter to Target Sequence

At least a 50:1 molar ratio of first adapter to cleavage ends wasdesired and an approximate ratio of 100:1 adapters to cleavage ends wastargeted. As λ DNA digested with EarI is known to result in 34 pairs ofcleavage ends, a 3400:1 mole ratio of adapters to λ DNA was used.

In 11 μl total reaction mixture, the following were combined, 5 μl fromthe fill-in reaction (approx. 40 nmoles target DNA), 4 μl of 5 μM firstadapter (2 μM final concentration), 1.1 μl 10× ligation buffer (1× finalconcentration), and 1 μl of T4. DNA ligase (400 units finalconcentration).

The reaction was incubated at 25° C. for 2 hours, then incubated at 75°C. for 10 minutes to inactivate the ligase as well as dissociateunligated adapter strand.

5. Second Klenow Fill-in Reaction

Filling in the single stranded portion of the target sequence/firstadapter created by dissociation of the unligated strand in step 4 above,was accomplished using the Klenow fragment DNA polymerase.

In 14 μl total was added 11 μl of DNA to which the first adapter hadbeen ligated (approx. 34.4 nM total adapted ends), 1.5 μl 10× Klenowbuffer (1×), 1.5 μl 2 mM dNTPs (50 μM each dNTP) and 0.05 μl Klenowfragment (0.25 units). This mixture was incubated at 37° C. for 30minutes, then heated to 75° C. for 10 minutes. Again, similar resultswere obtained using E. coli DNA polymerase.

6. Second Digestion with HgaI

To the 14 μl reaction mixture of step 6 was added 1 μl of HgaI (2units). The reaction was incubated at 25° C. for 3 hours. 1.6 μl of 5 MNaCl (0.5 M) was then added to raise the melting point of the targetsequence to above 70° C. The is reaction mixture was then heated to 65°C. for 20 minutes.

7. Ligation of Second Adapter to Target Sequence

The 16 μl reaction mixture from step 7 is expected to have anapproximate concentration of 4.4 nM target sequence with compatible endsfor the second ligation. This number is halved from the expectedconcentration of total target sequence. This was to account for theblunt end ligation of adapter 1 in the reverse orientation such thatHgaI cleavage would not occur.

To the 16 μl reaction mixture from step 7, was added 5 μl of 3 μM secondadapter prepared in step 3, above (0.3 μM), 5 μl 10× ligation buffer(1×), 23.5 μl water and 0.5 μl T4 DNA ligase (200 units). The reactionmixture was incubated at 37° C. for 30 minutes then heated to 65° C. for10 minutes.

8. PCR Amplification

5 μl of the captured target sequence from step 7 is used as the templatefor PCR amplification (approx. 440 μM total; 14.7 μM each end). To thiswas added 1.25 μl each of 10 μM T7 primer, and 10 μM T3 primer (0.25 mMprimer), 5 μl 10×PCR buffer (1×), 5 μl 4×2 mM dNTPs (200 μM each dNTP),24.5 μl water and 0.5 μl Taq polymerase (2.5 units).

PCR was carried out for 40 cycles of 94° C. for 30 seconds, 55° C. for30 seconds and 72° C. for 30 seconds. Controls were run using water, λDNA cut with EarI and uncut λ DNA subjected to steps 1-7. 2 μl from thereaction was run on a 4% NuSieve® Agarose gel, indicating a 62-bpamplicon which is carried into the next step.

9. Labeling Asymmetric PCR

The 62-bp amplicon produced in step 8 is next labeled with a 5′-F labelby asymmetric PCR.

44 μl of the PCR amplicon from step 8 (50 fmoles) is mixed with 5 μl of10 μM T7-5′F primer (1 μM primer), 2 μl of 10×PCR buffer (1× buffer), 3μl of 100 mM MgCl₂ (5 mM), 5 μl of 4×2 mM dNTPs (200 mM each dNTP) and0.5 μl Taq polymerase (2.5 units).

PCR was carried out for 40 cycles as described in step 8. 3 μl from thisreaction was the run on 4% NuSieve® Agarose gel and compared to theamplicon from step 8 to confirm florescent labelling.

9. Results

The florescent captured sequence was heated to 95° C. briefly, thenbuffered with 6×SSPE, 10 mM CTAB and 0.2% Triton X-100. The capturedsequence was then probed on an oligonucleotide array having thecombinatorial array shown in FIG. 4. FIG. 5, panel A shows the expectedhybridization pattern of λ DNA to the array of FIG. 4 as denoted by theblackened regions on the array. FIG. 5, panel B illustrates the actualhybridization pattern of captured Type-IIs sites from λ DNA on an arrayas shown in FIG. 4. The close correlation between expected and actualhybridization is evident.

Example 2

The above capture methods were applied to a genomic library of 12 knowncosmids from yeast chromosome IV. The clones have been previouslyphysically mapped using EcoRI-HindIII fragmentation. The specificlibrary, including known map positions and overlap of the 12 cosmids, isillustrated in FIG. 6.

The twelve genomic clones were constructed in a pHC79 vector, in E. colihost HB101. Cosmid DNA was prepared from 3 ml cultures by an alkalinelysis miniprep method. The miniprep DNA was digested with EcoRI andHindIII to confirm the known fingerprint of the large cloned inserts.Cosmid DNA was treated with linear DNAase, Plasmid-Safe™ DNAse, at 37°C. for 15 minutes, followed by heat inactivation. The DNAse treatmentwas carried out to remove any potential spurious EarI digested sitesresulting from contaminating bacterial DNA. This leaves cosmid DNAsubstantially untouched. After confirming the presence of clean bandingcosmid DNA, the resulting cosmids were then subjected to the capturemethods described above. The pCH79 vector, without a yeast insert, wastransformed into HB101 and isolated as a miniprep, to serve as acontrol.

The data from the array was normalized as follows. First, the probearray was normalized for background intensity by subtracting thebackground scan (hybridization buffer with no target). Second, the datawas normalized to the specific vector used in producing the cosmids.Normalization to the vector had two parts: first the average intensityof four hybridizing markers present in pHC79 vector was calculated foreach scan, for use as an internal control in that scan. This intensitywas divided into all intensities in that scan, and second the overallbackground intensity of the pCH79 vector in a bacterial host, absent ayeast insert, was subtracted. The array signal was normalized forrelative hybridization of the probes on the array, by using equimolartarget mixtures for each probe. Finally, the four values correspondingto the pCH79 markers were discarded:

The resulting hybridization patterns were then correlated, pair-wise,between all cosmids. Specifically, the signal intensity for each probewas compared among the same probe's intensity for all other fragments.Where the signals were the same, there was some correlation. The moresignals that were the same, the higher the correlation score.

These correlation scores are plotted against the known percent overlapfor these cosmids as determined from the EcoRI/HindIII physical map.This plot is shown in FIG. 7A. As is apparent, the correlation ofhybridization scores between fragments is readily correlatable topercent overlap of the fragments.

Example 3 Simulated Annealing

The correlation scores from yeast chromosome IV, above, were used toconstruct a best fitting contig, using the simulated annealing processas described by Cuticchia et al., The use of simulated annealingin'chromosome reconstruction experiments based on binary scoring,Genetics (1992) 132:591-601. A global maximum was sought for the sum ofcorrelation coefficient scores for a given sequence of cosmids in therandomly constructed and permutated contig. The resulting high scoringcontigs for all 12 cosmids and for the 10 “strong-signal” cosmids areshown below. Each cosmid was assigned a rank based upon the knownposition of that cosmid, and these are as follows:

TABLE 1 Cosmid Number Cosmid Rank 9371 A 8552 B 8087 1 9481 2 9858 39583 4 8024 5 8253 6 9509 7 9460 8 8064 9 9831 10 

Simulated, annealing of all twelve cosmids produced the followingordering:

-   -   (1 2 3 4) (7 6 5) A B (8 9 10)

Inclusion of the weaker signal cosmids, A and B, results in someshuffling of the predicted order of the cosmids. Removal of cosmids Aand B, the “weak-signal” cosmids, produced the following ordered map ofthe remaining ten cosmids:

-   -   (1 2 3 4 5 6 7) (8 9 10) which reflects the proper ordering and        indicates the existence of the two “islands” of cosmids as seen        in the physical map.

As can be seen, the inclusion of the weaker signal cosmids A and B, 8552and 9731, inverts the order of clones in the center positions (5, 6 and7), and improperly'places

Example 4 Simulated Mapping of Yeast Chromosome III

To determine how well the distribution of points in FIG. 7A matches thedistribution of scores expected for a random set of yeast cosmids, arandom set of fifty 35 to 40 kb sequences from yeast chromosome III(“YCIII”) were simulated. A list of perfect matches corresponding toEarI associated tetramers was also generated. Due to the difficulty inassigning simulated intensity scores for these markers, the markerprobes were scored as 1, and 0 for non marker probes. Inner productscores were used instead of correlation coefficients to determine thesimilarity of the marker sets in 1225 comparisons of the fifty simulatedYCIII cosmids. The scores were plotted against expected overlap, andthis is shown in FIG. 7B. Even when perfect information regarding markeridentities in the tetramer sets is compared, a certain amount of scatteris seen in the plot. Additionally, comparison of sequences with nooverlap generate inner product scores ranging from 0.05 to 0.4. Thesetwo features are characteristic of the actual data shown in FIG. 7A.

The simulation was repeated using BbsI and HphI as the first cleavingenzyme, and the results are shown in FIGS. 7C and 7D, respectively. Fromthis data, it can be seen that the amount of scatter in a particularplot is a function of the inverse of the frequency of cleavage sites(e.g., number of markers) in the target sequence. In particular, using.HphI as the first cleaving enzyme would produce 564 markers in YCIII,whereas BbsI would yield 212 and EarI would yield 274. The scatter forthe more frequently cutting HphI enzyme is substantially less than thatfor BbsI and EarI. Additionally, as noted previously; the Y intercept isalso affected by the number of markers in the target sequence, as wellas the frequency of a particular marker (e.g., marker duplication). Bothof these factors may be influenced by the choice of capture methods andenzymes.

The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents. Allpublications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

1. A method of identifying sequences in a polynucleotide sequence,comprising: first cleaving the polynucleotide sequence with a firsttype-IIs endonuclease; first ligating a first adapter sequence to thepolynucleotide sequence cleaved in said first cleaving step, said firstadapter having a recognition site for a second type-IIs endonuclease;second cleaving the polynucleotide sequence resulting from said firstligating step, with the second type-IIs endonuclease; second ligating asecond adapter sequence to the polynucleotide sequence cleaved in saidsecond cleaving step; and determining the sequence of nucleotides of thepolynucleotide sequence between the first and second adapter sequences.2. The method of claim 1, wherein: in said first cleaving step, thefirst type-IIs endonuclease is selected from the group consisting ofBsmAI, EarI, MnlI, PleI, AlwI, BbsI, BsaI, BspMI, Esp3I, HgaI, SapI,SfaNI, BseRI, HphI and MboII; and in said second cleaving step, thesecond type-IIs endonuclease is selected from the group consisting ofHgaI, BbvI, BspMI, BsmFI and FokI.
 3. The method of claim 2, wherein insaid first cleaving step, the first type-IIs endonuclease is EarI; andin said second cleaving step, the second type-IIs endonuclease is HgaI.4. The method of claim 1, wherein in said first and second ligatingsteps, said first and second adapter sequences comprise primersequences.
 5. The method of claim 4, wherein prior to said determiningstep, the sequence of oligonucleotides in the polynucleotide between thefirst and second adapter sequences is amplified.
 6. The method of claim1, wherein in said determining step, the sequence of nucleotides betweenthe first and second adapter sequences is determined by hybridization toan oligonucleotide probe.
 7. The method of claim 6, wherein saidoligonucleotide probe is a positionally distinct probe on anoligonucleotide array, a position of the probe being indicative of thesequence of the probe.
 8. A method of generating an ordered map of alibrary of genomic fragments, the method comprising: identifyingsequences in each of the genomic fragments in the library, according tothe method of claim 1; comparing the sequences identified in eachfragment with the sequences identified in each other fragment to obtaina level of correlation between each fragment and each other fragment;and ordering the fragments according to their level of correlation.
 9. Amethod of identifying polymorphisms in a target polynucleotide sequence,the method comprising: identifying sequences in a wild-typepolynucleotide sequence, according to the method of claim 1, repeatingsaid identifying step on the target polynucleotide sequence; anddetermining differences in the sequences identified in each of saididentifying steps, the differences being indicative of a polymorphism.10. The method of claim 1, wherein said sequences in a polynucleotidesequence are proximal to a polymorphism.
 11. A method of identifying asource of a biological sample, the method comprising: identifying aplurality of sequences in a polynucleotide sequence derived from thesample, according to the method of claim 1; and comparing the pluralityof sequences identified in said identifying step with a plurality ofsequences identically identified from a polynucleotide derived from aknown source, identity of the plurality of sequences identified from thesample with the plurality of sequences identified from the known sourcebeing indicative that the sample was derived from the known source. 12.A method of determining a relative location of a target nucleotidesequence on a polynucleotide, the method comprising: generating anordered map of the polynucleotide according to the method of claim 8;fragmenting the polynucleotide; determining which fragment includes thetarget nucleotide sequence; correlating a marker on the fragment with amarker on the ordered map to identify the approximate location of thetarget nucleotide sequence on the polynucleotide.